Many drugs are absorbed by passive diffusion through a hydrophobic cellular membrane, which does not participate in the absorption process. The amount of absorbed drug is controlled by two processes. First, a high concentration of the active substance at the membrane surface will enhance cellular absorption (Fick's Law). Since cells function in an aqueous environment, enhancing the water solubility of a drug increases its concentration at the locus of absorption. However, while greater water solubility may be expected to enhance the bioavailability of drugs, this is frequently not the case due to a second, competing process that affects the overall absorption process. The absorptive cell membrane is composed mainly of lipids that prevent the passage of hydrophilic compounds, but which are highly permeable to lipid soluble substances. Therefore, the design of bio-available drugs must balance two opposing forces. On the one hand, a drug that is very hydrophilic may have a high concentration at the cell surface but be impermeable to the lipid membrane. On the other hand, a hydrophobic drug that may easily “dissolve” in the membrane lipids may be virtually insoluble in water producing a very low concentration of the active substance at the cell surface.
To circumvent these problems, a number of strategies have been used to maintain the hydrophobicity of the drug and at the same time to provide a “packaging” matrix that increases its aqueous concentration. For example, emulsions can be prepared for the parenteral delivery of drugs dissolved in vegetable oil [Collins-Gold, L., Feichtinger, N. & Warnheim, T. (2000) “Are lipid emulsions the drug delivery solution?” Modern Drug Discovery, 3, 44-46.] Alternatively, artificial membranes of liposomes have been used to encapsulate a variety of drugs for different delivery routes, including oral, parenteral and transdermal [Cevc, G and Paltauf, F., eds., “Phospholipids: Characterization, Metabolism, and Novel Biological Applications”, pp. 67-79, 126-133, AOCS Press, Champaign, Ill., 1995]. All these methods require amphiphiles, compounds that have a hydrophilic or polar end and a hydrophobic or nonpolar end, such as phospholipid, cholesterol or glycolipid or a number of food-grade emulsifiers or surfactants.
When amphiphiles are added to water, they form lipid bilayer structures (liposomes) that contain an aqueous core surrounded by a hydrophobic membrane. This novel structure can deliver water insoluble drugs that are “dissolved” in its hydrophobic membrane or, alternatively, water soluble drugs can be encapsulated within its aqueous core. This strategy has been employed in a number of fields. For example, liposomes have been used as drug carriers since they are rapidly taken up by the cells and, moreover, by the addition of specific molecules to the liposomal surface they can be targeted to certain cell types or organs, an approach that is typically used for drugs that are encapsulated in the aqueous core. For cosmetic applications, phospholipids and lipid substances are dissolved in organic solvent and, with solvent removal, the resulting solid may be partially hydrated with water and oil to form a cosmetic cream or drug-containing ointment. Finally, liposomes have been found to stabilize certain food ingredients, such as omega-3 fatty acid-containing fish oils to reduce oxidation and rancidity (Haynes et al, U.S. Pat. No. 5,139,803).
In an early description of liposome formulation (Bangham et al., 1965 J. Mol. Biol. 13, 238-252), multilammelar vesicles were prepared by the addition of water and mechanical energy to the waxy film that was formed by removing the organic solvent that was used to dissolve the phospholipids. In later work, it was found that the combination of sterols (cholesterol, phytosterols) and phospholipids allowed the formulation of liposomes with more desirable properties, such as enhanced stabilization and encapsulation efficiency. The patent and scientific literature describe many methodological improvements to this general strategy. However, none presently known achieves the efficient delivery rates of the present invention.
Even though liposomes provide an elegant method for drug delivery, their use has been limited by cumbersome preparation methods and the inherent instability of aqueous preparations. A number of patents describe the large scale preparation of pre-liposomal components that can be hydrated later to form the desired aqueous-based delivery vehicle. Evans et al. (U.S. Pat. No. 4,311,712) teach that all the components (phospholipids, cholesterol and biological agent) can be mixed in an organic solvent with a melting point near that of room temperature. After solvent removal by lyophilization, addition of water produced liposomes with the biologically active material “dissolved” in the membrane. Similarly, U.S. Pat. No. 5,202,126 (Perrier et al.) teaches the addition of all the components in the organic phase, but with solvent removal accomplished by atomization following the method described by Redziniak et al. (U.S. Pat. Nos. 4,508,703 and 4,621,023). The pulverulent solid so produced can then by hydrated, homogenized and converted into a cream for the typical delivery of the biologically active material, in this case pregnenolone of pregnenolone ester. Orthoefer describes the preparation of liquid crystal phospholipids (U.S. Pat. No. 6,312,703) as a novel carrier for biologically active compounds. In this method, the various solid components are pre-mixed and then subjected to high pressure to form a lecithin bar that can be used in cosmetic applications as soap or the pressurized components can be extruded as a rope and cut into pharmaceutical-containing tablets. Unlike previous work, this method does not teach premixing in organic solvent or homogenization in water.
The utility of a dried preparation to enhance the stability and shelf life of the liposome components has long been recognized, and numerous methods have been devised to maintain the stability of liposomal preparations under drying conditions. Schneider (U.S. Pat. No. 4,229,360) describes the preparation of encapsulated insulin in liposomes by adding the aqueous peptide solution to a film of phospholipids. Lyophilization of this liposomal mixture in the presence of gum Arabic or dextran produced a solid that could be reconstituted with water to form liposomes. However, following a similar procedure to encapsulate cyclosporine, Rahman et al. (U.S. Pat. No. 4,963,362) teach that the lyophilization step can be performed without the addition of other additives, such that the re-hydrated liposomes maintain their ability to encapsulate the bioactive substance. Vanlerberghe et al. (U.S. Pat. No. 4,247,411) teach a similar process, but include sterols with the phospholipids to provide a more stable liposome. In an effort to enhance the stability and dispersibility of liposomes in a solid matrix, Payne et al. (U.S. Pat. Nos. 4,744,989 and 4,830,858) describe methods for coating a water soluble carrier, such as dextrose, with a thin film of liposome components. When added to water, the carrier dissolves and the liposome components hydrate to form liposomes.
The goal of all these methods is to produce a solid that can be re-hydrated at a later time to form liposomes that can deliver a biologically active substance to a target tissue or organ. Surprisingly, there have been only two reports that use the dried liposome preparations themselves, with no intermediate hydration, as the delivery system. Ostlund, U.S. Pat. No. 5,932,562 teaches the preparation of solid mixes of plant sterols for the reduction of cholesterol absorption. Plant sterols or plant stanols are premixed with lecithin or other amphiphiles in organic solvent, the solvent removed and the solid added back to water and homogenized. The emulsified solution is dried and dispersed in foods or compressed into tablets or capsules. In this case, the active substance is one of the structural components of the liposome itself (plant sterol) and no additional biologically active substance was added. Manzo et al. (U.S. Pat. No. 6,083,529) teach the preparation of a stable dry powder by spray drying an emulsified mixture of lecithin, starch and an anti-inflammatory agent. When applied to the skin, the biologically active moiety is released from the powder only in the presence of moisture. Neither Ostlund nor Manzo suggest or teach the use of sterol, and lecithin and a drug active, all combined with a non-polar solvent and then processed to provide a dried drug carrying liposome of enhanced delivery rates.
Substances other than lecithin have been used as dispersing agents. Following the same steps (dissolution in organic solvent, solvent removal, homogenization in water and spray drying) as those described in U.S. Pat. No. 5,932,562, Ostlund teaches that the surfactant sodium steroyl lactylate can be used in place of lecithin (U.S. Pat. No. 6,063,776) Burruano et al. (U.S. Pat. Nos. 6,054,144 and 6,110,502) describe a method of dispersing soy sterols and stanols or their organic acid esters in the presence of a mono-functional surfactant and a poly-functional surfactant without homogenization. The particle size of the solid plant-derived compounds is first reduced by milling and then mixed with the surfactants in water. This mixture is then spray dried to produce a solid that can be readily dispersed in water. Similarly, Bruce et al. (U.S. Pat. No. 6,242,001) describe the preparation of melts that contain plant sterols/stanols and a suitable hydrocarbon.
On cooling these solids can be milled and added to water to produce dispersible sterols. Importantly, none of these methods anticipate the type of delivery method described here as a means to delivery hydrophobic, biologically active compounds.
All of the above described art, either deals with lowering of cholesterol or with a variety of techniques used in an attempt to solubilize some hydrophobic drugs using specific lipids. None teach or suggest a generalized approach to enhance solubilization in a water environment and to enhance the rate of diffusion of hydrophobic drugs through lipid membranes of cell walls so that the drug has increased bio availability at any given dose.
The above described art describing solubilizing hydrophobic drugs is focused on creating an artificial membrane whose composition does not correspond to that found in natural, cellular structures. For example, previous preparations describe a mixture in which a sterol is incorporated in the phospholipid (amphiphile) phase at a weight ratio of between 1% and 20%, and preferably of 10% [Perrier et al., U.S. Pat. No. 5,202,126 (c2, line 33)], a prejudice that is re-affirmed in later work [Meybeck & Dumas, U.S. Pat. No. 5,290,562 (c3, line 29)]. While these ratios provide useful properties for the intended low drug loading use of this delivery system, they do not provide sufficient capacity for higher drug loading. In erythrocyte plasma membrane, liver plasma membrane or myelin, the ratio of cholesterol to phosphatidyl choline is close to one and the ratio of cholesterol to all membrane phospholipids is usually less than three. While the reason for these ratios is not known it is speculated that this combination stabilizes the membrane and allows highly hydrophobic proteins to span the lipid portion of the membrane. Based on these considerations from native cellular membranes, the present applicant explored the ratios set forth herein.
Further, in contrast to the above described art, this invention reveals that the ratio of the amphiphile to the drug and plant sterol combination also plays an unexpected role for the delivery of this class of drug substance. In order to form creams and parenteral liposomal preparations, previous work focused on the preparation of dispersions containing small liposomal particles (less than 1 μm) by maintaining a high ratio of lecithin to the other components. This prejudice is shown by the requirement that the sum of the drug and the sterol present should not exceed about 25% and preferably about 20% of the total lipid phase present. Hence, the previous art teaches a ratio of lecithin to the sum of the sterol and drug components of at least 3.0, and preferably 4.0 [Perrier et al., U.S. Pat. No. 5,202,126 (c2, line 45), Meybeck & Dumas, U.S. Pat. No. 5,290,562 (c3, line 29)]. While this preferred ratio of lecithin to the other components may be appropriate for certain delivery systems, such as creams, ointments and parenteral liposomal preparations, a simple calculation demonstrates its potential impracticality when applied to a conventional solid delivery system, such as certain food products, tablets and capsules. For example, if 100 mg of drug is required for an efficacious dose and if 100 mg of sterol is included, then according to the specifications, at least 800 mg of lecithin is needed to give a total mass of 1.0 gm. Excipients must then be added to provide a compressible and flowable powder matrix for compression or encapsulation. The resulting delivery system requires many tablets or capsules in order to deliver the active drug substance, leading to high costs and poor subject compliance. Thus, in practical terms, the previous method is severely limited and useful only for creams, ointments and parenteral liposome aqueous preparations or to solid formulations (tablets and capsules) of poorly soluble drugs that have very high potency and that can be delivered in very small doses.
In previous art, the selection of an acceptable ratio of ingredients was determined by the particle size of the dispersion after homogenization and the subsequent stability of that dispersion with time and temperature. As measured by the mean size and polydispersity, there is prejudice against those dispersions that change over time and that undergo sedimentation. Importantly, to maintain liposomal “quality,” the dispersion must be characterized by a small particle size to enhance the stability of the dispersion for intended uses [Perrier et al., U.S. Pat. No. 5,202,126 (c4, line 61)]. Departure from this preferred ratio produces sediment which “detracts from the stability of the liposomes”[Perrier et al., U.S. Pat. No. 5,202,126, (c5, line 10)]. Sediments, so formed, are considered sub-optimal and are discarded as inappropriate for further use.
An object of this invention is to enhance the usefulness of a hydrophobic drug substance by its combination with sterols such that the ratio of these two components to an amphiphile is chosen to produce liposomal particles greater than 1 μm. This combination, which produces a particle dispersion that would be characterized by previous criteria as low quality and of little usefulness, has surprising and unexpected utility. Thus, this combination produces a delivery system with the following useful, novel and unexpected advantages: (1) a dispersed solution that can be dried and re-hydrated to produce a dispersion of particles that is similar to the dispersion from which it was derived, without re-addition of energy; (2) high drug loading capacity by minimizing the amount of amphiphile in the mix; (3) a dispersed solution that is stable to conventional drying methods without the addition of large amounts of stabilizers. In addition, the dried solid so manufactured can be easily added to a food product or compacted in a tablet or capsule to render the hydrophobic drug bioavailable on ingestion and easily deliverable in a pharmaceutical format.
A dispersion consisting of large particles is consistent with currently held theories on the physical chemical and biochemical events that occur in fat digestion and provide a framework for understanding the basis for enhanced lipid drug absorption described here. Large emulsion particles containing a variety of fats, fat soluble vitamins and lipid nutrients enter the small intestine from the stomach. For example, the fat globules in bovine and human milk have diameters of 1-10 μm and 1.5-4 μm, respectively, similar in size to the phospholipid sterol drug dispersions described here, but larger than those described in previous patent applications (Patton & Keenan, Biochim Biophys Acta, 1975, 415: 273-309; Hamosh et al., Pediatrics, 1984, 75(suppl): 146-150). In the small intestinal lumen, the globule dispersions are exposed to bile salt, additional phospholipid and a variety of enzymes and proteins that serve to reduce the particle size in a controlled series of hydrolytic and physical chemical steps. In the presence of excess bile salt, small vesicles and/or liquid crystals are formed that become saturated with lipolytic products and fat soluble nutrients, which provides a thermodynamically favorable environment for maximum rates of lipid uptake (Thomson et al., Can J Physiol Pharmacol, 1989, 67: 179-191). Packaging a water insoluble drug in a sterol amphiphile matrix characterized as a dispersion of large particles may allow the same absorptive steps to occur that are used by naturally occurring lipids and nutrients found in the diet. Importantly, addition of small particles may not be compatible with one or more steps of this absorption process and may lead to less efficient uptake.